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Drug Induced Hemolytic Anemia (DIIHA)
Sandra J. Nance, MS, MT(ASCP)SBBSenior Director, IRL, American Red Cross
Adjunct Assistant Professor, University of PennsylvaniaSenior Director, American Rare Donor Program
Philadelphia, Pennsylvania, USA
Chilean Society of Hematology
XX Congress of the Chilean Society of Hematology
X Congress of Transfusion Medicine
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American Red Cross Headquarters
Washington, DC
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American Red Cross Immunohematology Reference Laboratories
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Disclaimers
Member of 2018 Grifols Standing Committee
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Abbreviations
• AIHA – Autoimmune Hemolytic Anemia• DAT – Direct Antiglobulin Test• DIIHA- Drug-Induced Immune Hemolytic Anemia• IRL – Immunohematology Reference Laboratory• NRLBGS – National Reference Laboratory for
Blood Group Serology• RBC – Red Blood Cells
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Drug-Induced Immune Hemolytic Anemia (DIIHA)
• First suspected in 1953 by Snapper et al (Ann Int Med, 619)
• First documented in 1956 by Harris (J Lab Clin Med, 760)
• By 1967, 34 published cases involving 15 drugs
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DIIHA Reports by G Garratty
Number of Drugs reported tocause DIIHA or positive DAT
• 1980 – 33 drugs
• 1989 – 70 drugs
• 2003 – 100 drugs
• 2014 – 138 drugs
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DIIHA Case Documentation
Some reports are not so well documented for the drug as causative agent, often reported as:
• Hemolytic Anemia starts after starting a drug
• Hemolytic Anemia resolves after stopping drug
• Positive DAT
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DIIHA Documentation
To prove that hemolytic anemia is due to drug:
• Drug required to be present in test media to demonstrate antibody reactivity
• Or, prove that hemolytic anemia was induced by drug with clinical documentation
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DIIHA Documentation
• How to prove that drug must be present in order for antibody to react
–Can be difficult to prove with testing
–Preferred to show antibody in eluate
–Antibody in serum supports but does not prove as drug antibodies have been detected in “normal” donors and patients
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DIIHA Information
• Why would drug antibody be in serum of healthy person?
–May be present due to past exposure
–May be a feature of the particular drug –cells coated with cephalosporins non-specifically take up antibody
• Some drugs do not require presence of drug to react – look like autoantibody
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DIIHA - How to Prove?
• Proof Positive: Clinical and Serologic evidence
–Patient has clinical evidence of hemolysis
– Stop the drug
–Observe that Hemolytic Anemia stops, DAT becomes negative and no eluate reactivity
– Start the drug
–Observe that Hemolytic Anemia and serologic evidence is present
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DIIHA - How to Prove?
• Proof Positive: Clinical and Serologic evidence
–Patient has clinical evidence of hemolysis
– Stop the drug
–Observe that Hemolytic Anemia stops, DAT becomes negative and no eluate reactivity
– Start the drug
–Observe that Hemolytic Anemia and serologic evidence is present
RARELY DONE ON PURPOSE –
SOMETIMES DONE BY ACCIDENT
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Proposed Mechanisms for
From Petz and Garratty Acquired Immune Hemolytic Anemias, Churchill Livingstone NY 1990
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Another way to think about this
Cambridge University Press
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Technical aspects
Two ways that drug antibody investigations originate
–Clinically requested - Specific request from patient’s physician suspecting DIIHA due to clinical symptoms and laboratory results
– Laboratory discovery• Hemolysis in serum tube• Positive DAT, negative eluate• Positive antibody screen with all cells
reactive
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Indications for DIIHA Testing if Laboratory Discovered• Positive antibody screen (variable)
– Could be positive if drug currently being administered
– Could be negative if antibody is all on patient’s RBCs
• If autocontrol tested with screen or panel, expected to be positive (or DAT positive)
• If Eluate tested – expected to be negative which is the clue that positive DAT is not due to warm AIHA
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Pertinent Facts of DIIHA Samples
• Reactivity variable generally according to sensitivity of method, except for Solid Phase
– Gel, Ficin, PEG IAT> SPRCA, Saline IAT> 37C or IS
• Reactivity wanes as time elapsed since drug administered
• Some cases reported blood group specificities
• Normal donors and patients may have drug antibodies
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DIIHA Antibodies with Blood Group Specificity
Antibody Specificity Drug Implicated
Anti-e or relative anti-e Diclofenac, Latanoxef,
Nabumetone, Piperacillin,
Teniposide, Tolmetin,
Anti-C Rifampicin
Anti-E Nomifensine
Anti-f Cefotetan, Sulindac
Anti-Jka Chorpropamide
Anti-H Sulfamethoxazole
Anti-Rh17 Sulindac
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Normal Sera may have Drug Antibodies
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DIIHA – Four Categories
• Drug dependent antibodies – Penicillin type
– Detection method – drug coated cells
• Drug dependent antibodies – non-penicillin type
– Detection method – drug solution addition
• Nonimmunologic adsorption of proteins onto RBCs
– Detection method – compare with normal serum, difficult to prove
• Drug independent antibodies – autoantibodies
– Detection method – routine antibody screen, eluate
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DIIHA
DRUG DEPENDENT ANTIBODIES
PENICILLIN TYPE
(DRUG COATED RBCs)
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DIIHA - Drug dependent antibodies
• Penicillin type – Ampicillin, amoxicillin, piperacillin, ticarcillin (cross react with
Penicillin)
– Methicillin, nafcillin (do not cross react with Penicillin)
• Detection method – Drug coated cells
• Drugs in this category include:– Carbromal Erythromicin Tolbutamide
– Cefotaxime Isoniazid Azopropazone
– Cefotetan 6-mercaptopurine Carbimazole
– Cefoxitin Penicillins Cephalothin
– Cisplatin Streptomicin Cianidanol
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DIIHA – Drug Coated Cells
• Commonly, antibodies to Penicillin can be detected with drug-coated cells
• Normal people can have antibody to penicillin
• Most patients who have penicillin antibody also have antibody to the b-lactam nucleus thus are cross reactive with semi-synthetic penicillins that have different side chains but still have the b-lactam nucleus
• Some patients have antibody to the side chains only and thus do not cross-react with other penicillins
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DIIHA – Drug Coating Method
• Prepare drug coated cells using appropriate drug solution (Ag negative if needed)
• Test coated and uncoated RBCs in parallel with serum and eluate of patient, and normal inert serum.
• If normal serum is positive, dilute normal serum and patient serum 1:20 or 1:100 for cefotetan
• Test– 2 drops serum/eluate and 1 drop 4% RBCs– 37C 60 minutes– Wash, add AHG, read macro and micro
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DIIHA – Drug Hapten Inhibition
Test set of titration tubes:– 0.1 mL serum dilution– 0.1 mL drug solution
• Control set of tubes:– 0.1 mL serum dilution– 0.1 mL PBS
• 37C, 60 minutes• Add drug coated RBCs• 37C, 60 minutes, read• Wash, add AHG, Read
Prepare:• Titer patient’s serum
versus drug coated RBCs• Prepare Inhibition solution
(10mg/mL)• Prepare master dilution of
patient’s serum one tube past titer end-point
• Prepare 2 sets of tubes, “test” and “control”
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DIIHA – Drug Hapten Inhibition
• Interpretation
– Serum + Drug solution same titer as PBS
• No inhibition- antibody not inhibited by drug
– Serum + Drug solution lower titer than PBS
• Inhibition – proving drug antibody present
– Serum + Drug solution higher titer than PBS
• Drug antibody reacts preferentially by Drug Solution Addition method
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DIIHA
DRUG DEPENDENT ANTIBODIES
NON-PENICILLIN TYPE
(DRUG SOLUTION ADDITION)
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DIIHA - Drug Dependent Antibodies –Non Penicillin Type
• Detection method – drug solution addition
– “Immune Complex”
• Drugs in this category include:
– Carbimazole Carboplatin Latamoxef
– Tolmetin Cefotetan Diclofenac
– Cefotaxime Teniposide Zomepirac
– Cetazidime Glafenine
– Cianidanol Nomifensine
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DIIHA Drug Solution Addition Method(also called “Immune Complex”)
• Most drugs are not bound to RBCs and cannot be prepared in vitro
• Not well understood how they act in vivo to cause hemolytic anemia
• Thought to cause Hemolytic Anemia by activating complement
• Small amount of drug needed
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Drug Solution Addition Type
• Acute complement-mediated hemolysis–Up to 50% have renal failure
• C3 is present on RBCs, but IgG and IgMcan also be present
• Antibodies are often IgM, but can be IgG• In vitro, in presence of drug, can see
hemolysis, agglutination and sensitization• Stop drug, hemolytic anemia resolves
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DIIHA – Drug Solution Addition MethodPrepare Drug Solution (1mg/mL) and test tubes as follows:
Serum Drug Solution
Fresh Complement
PBS
2 drops 2drops ----- -----
2 drops 2 drops 2 drops -----
2 drops ----- 2 drops -----
2 drops ----- ----- 2 drops
----- 2 drops 2 drops -----
----- ----- 2 drops 2 drops
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DIIHA – Drug Solution Addition MethodPrepare Drug Solution (1mg/mL) and test tubes as follows:
Serum Drug Solution
Fresh Complement
PBS
2 drops 2drops ----- -----
2 drops 2 drops 2 drops -----
2 drops ----- 2 drops -----
2 drops ----- ----- 2 drops
----- 2 drops 2 drops -----
----- ----- 2 drops 2 drops
Test Controls
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DIIHA – Drug Solution Addition Method
• Prepare two sets of tests, one with untreated and one with enzyme treated RBCs, 6% -10% suspension
• 37C, 60 minutes incubation
• Read for hemolysis and agglutination
• Wash, add Polyspecific AHG, read
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DIIHA
NONIMMUNOLOGIC ADSORPTION OF
PROTEINS ONTO RBCs
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DIIHA -Nonimmunologic Adsorption of Proteins onto RBCs• Detection method:
– Comparison to normal serum– Clinical/temporal relationship to hemolytic anemia
• Drugs in this category include:– Cephalosporins– Suramin– Diglycoaldehyde (INOX)– Cisplatin/Oxaliplatin– Tazobactam (in Zosyn)– Sulbactam (in Unasyn)– Clavulanate (Augmentin and Timetin)
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DIIHA Non-immunologic Adsorption of Protein on RBCs
• Proteins adsorb to RBCs causing positive DAT• Detected because normal plasmas react with
RBCs incubated with drug, but eluates from those RBCs are negative
• AHG tests with anti-albumin standardized for use with RBCs are positive
• Different sources of AHG may or may not contain anti-albumin, thus detection may be variable
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DIIHA Non-immunologic Adsorption of Protein on RBCs
• Positive DAT may be due to IgG, IgM, IgA, C3, or Albumin
• Eluate from patient’s RBCs is negative with drug-coated or uncoated RBCs, even in presence of drug
• Patient’s serum and normal donor serum may react with drug coated but not with untreated RBCs, Patient’s serum may be stronger
• Patients may have +DAT but no hemolytic anemia
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DIIHA
DRUG INDEPENDENT ANTIBODIES
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DIIHADrug Independent Antibodies
• Autoantibodies
– Detection method – routine antibody screen, eluate
• Drugs in this category include:
– Methyldopa - Cladrabine
– Levodopa - Cianidanol
– Procainamide - And others reactive by this
– Mefenamic acid and other mechanisms
– Fludarabine
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DIIHA - Drug Independent AntibodiesDrug Induced Autoantibodies
• Antibodies do not require drug to be present• Serologic and clinical features similar to idiopathic AIHA• Methyldopa first and best studied
– Positive DAT post therapy 3-6 mo, hemolytic anemia rarely before 18 weeks
– No anamnestic response upon re-administration– Up to 15% on drug make autoantibodies– Less than 0.5% with positive DAT have DIIHA– Commonly have Rh specificity– If have DIIHA and removed from drug, positive DAT
can persist for 2 years
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DIIHA
CEPHALOSPORINS
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DIIHA - Cephalosporins
• Pharmacologic Aspects:
– Most administered by injection
• Plasma half life of 1-2 hours
• Metabolites not common
– Most orally administered are less of a risk for
DIHA due to relatively lower dose
Cephalexin, Cefadroxil, Cefaclor, Cefuroxime Axetil, Cefixime, Cephradine
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DIIHA Cephalosporins - Viraraghaven et al,
• US- FDA reviewed 85 cases of HA associated with
Cefotetan
– 5 were published
– +DAT in 50 of 52 tested
– Cefotetan antibodies in 30 of 30 tested
– Renal dysfunction in 7 patients
– 15 fatalities
– Prev. administered Cefotetan – 15 (4 died)
– 32 for treatment, 47 prophylaxis
Adv Drug React Toxicol Rev 2002;21:101-107
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DIIHA – Cephalosporins
• Cefotetan data from multiple references (n=31)– 80% for surgery, one single 2 gm dose used– HA evident 1-13 days after drug administered– Nadir Hb S/P drug – mean 4.8 g/dL– Fatal HA 19% – Renal Failure 19%– All had Cefotetan Ab vs drug coated RBCs (median
titer 512, normal less than 20)– 30/31 positive with drug solution added– 40% reacted with RBCs without drug present
Petz and Garratty, Immune Hemolytic Anemias, p 296
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Summary – Investigation of DIHA
• DIIHA is rare
• If have positive DAT and negative eluate, more common to have anti-A or anti-B coating the RBCs
• 80% of all positive DATs yield negative eluates
• Investigate when evidence of Hemolytic Anemia and treatment with a drug in same time frame
• If drug never reported, use both serum and eluateand test with Drug coated and Drug solution method
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Drugs Implicatedin DIIHA
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Data on Drug AntibodiesDr Garratty’s Laboratory
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